Abstract/Project Summary Bladder cancer is the 6th most common cancer in the US with 81,000 new cases and 18,000 cancer related deaths each year. Patients with non-muscle invasive bladder cancer (NMIBC), the most common form of bladder cancer, are treated with tumor resection followed by intravesical (into the bladder) immunotherapy with the live mycobacterium Bacille Calmette Guerin (BCG). This has been the standard of care for over 40 years. However, tumors frequently recur, and up to 40% of patients are unresponsive to BCG therapy. Currently, BCG refractory disease has no effective alternative therapy and high risk NMIBC patients undergo radical cystectomy if they do not respond to BCG, severely impacting patient quality of life. In addition, there is currently a global shortage of BCG leading patients to receive suboptimal treatment regimens. Thus, alternative immunotherapy strategies to fight bladder cancer are warranted. Immunotherapies can be generalized to 2 classes, those that ?release the brakes? or ?push on the accelerator? of the immune system. Although NMIBC has a long history of treatment with BCG immunotherapy, comprehensive immune profiling of the bladder is still currently lacking, especially with respect to growing knowledge of T cell and dendritic cell (DC) subsets that mediate anti-tumor immunity. This proposal will, define the immune contexture of mouse and human bladder cancer and investigate novel immunotherapy strategies for bladder cancer in pre-clinical humanized mouse models with the aim of identifying regimens that can outperform or synergize with BCG therapy. Agonistic antibodies targeting CD40 are one promising approach to ?accelerate? anti-tumor immunity, however they have not been investigated within the bladder. CD40 agonism via antibodies requires engagement of FcyRIIB for efficient CD40 crosslinking, and Fc- engineered CD40 agonistic antibodies demonstrate enhanced therapeutic activity. This proposal will determine the therapeutic efficacy of a fully human, Fc-engineered, intravesically instilled CD40 agonist antibody in single agent and combination therapy regimens. Initial results using mouse models show that immune infiltrates in the tumor bearing bladder express CD40 principally in DCs but are not present in normal bladder, and intravesical delivery of CD40 agonist is safe and reprograms the bladder immune microenvironment towards anti-tumor immunity. The bladder tumor microenvironment also contains numerous exhausted T cells that could be targeted by checkpoint blockade immunotherapy. Further experiments will evaluate efficacy of CD40 agonism in combination treatment regimens with BCG or immune checkpoint blockade, and in BCG refractory disease. These studies will provide key information on the immune environment of bladder cancer and form a basis for translating CD40 agonism as a treatment approach for NMIBC. The training environment and resources available from the Ravetch Laboratory, a leader in the field of antibody therapeutics, at the Rockefeller University provide the ideal setting to conduct these studies.